Comment on “DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction”

Abstract

Metrics & MoreArticle Recommendations S tudies have demonstrated that RNAs can be 5′-capped with metabolites, including dephospho-CoenzymeA (dpCoA), but identification of the capped RNAs is difficult. Recently, Shao et al. 1 developed a new method for detection of 5′dpCoAcapped RNA based on the reaction with maleimide linked to a oligonucleotide sequence tag and subsequent nanopore sequencing (dpCoA tagseq). The method was applied to RNA isolated from mouse liver, thereby leading to the identification 44 genes producing 5′dpCoA-capped RNA. However, closer inspection revealed that many tagged reads were mapped to multiple sequences and predominantly to repetitive sequences, especially LSU and SSU rRNA derived sequences and other noncoding ncRNAs such as 7SL, 7SK, U1, and U6 sRNAs. In addition, in most cases, the mapped sequences were not located 3′ to the tag sequence as would be expected if a 5′ cap had reacted with the sequence tag. Instead, most of the reads, in addition to the mapped sequences, contained 5′ sequences derived from a control 5′dpCoA-capped RNA, which was spiked into the mouse RNA by the authors. These observations demonstrate that the reported 5′dpCoA-capped mouse liver RNAs are false positives and pinpoint potential pitfalls in the analysis of nanopore derived tagseq data.More than a decade ago, dephospho-CoA (dpCoA), Acetyl-dpCoA, Propionyl-dpCoA, and Succinyl-CoA were surprisingly identified as 5′ caps on RNA in E. coli based on mass

If the 5’ cap fits (wear it) – Non-canonical RNA capping

“…Given that dpCoA has a reactive thiol group, it is an ideal candidate for chemical conjugation using maleimides [ 107 ]. This led to the development of dpCoA tagSeq, which uses a maleimide-modified RNA to “tag” the dpCoA-RNA at the 5' end with a barcode sequence.…”